Advanced metastatic cancers are largely incurable since cancers have found multiple different ways to usurp signalling pathways to gain a growth advantage. Therefore it is unlikely that pharmacological attack on a single molecular target will significantly impact the long-term progression of the malignancy (Jones et al., 2008, Science 321: 1801-1806). Moreover, tumor cells become very heterogeneous as they evolve under the selective pressure of their microenvironment (Subarsky, P and Hill, R P, 2003, Clin Exp Metastasis 20: 237-250).
While our immune system has the capacity to rapidly respond and has the potential to recognize the antigenic variations presented by tumor cells (Cheever et al., 2009, Clin Cancer Res 15: 5323-5337), in particular advanced tumors are highly immunosuppressive. The ability to create an immunosuppressive environment permits the cancer cells to avoid detection by the immune system. They inhibit the maturation of local professional antigen-presenting cells by secreting cytokines and other molecules that inhibit the expression of costimulatory molecules, essential for the expansion of T cells (Strobl, H and Knapp, W., 1999, Microbes Infect 1: 1283-1299; Fiorentino, D F et al., 1991, J Immunol 146: 3444-3451). Tumors also directly inhibit T cells and instead of costimulatory molecules, many tumors express coinhibitory molecules. Some tumors may not express inhibitory molecules themselves, but recruit inhibitory cell types such as T-regulatory cells do. The fact that advanced tumors are highly immunosuppressive demonstrates the importance of the immune system in this context. Besides creating an immunosuppressive environment, tumor cells escape the immune system by poor or even lack of presentation of tumor antigens to effector T cells (Maeurer, M J, et al., 1996, Clin Cancer Res 2: 641-652).
All these properties make cancer a complex disease and a challenging one to treat.
Viruses have two important properties to overcome both heterogeneity and immune escape mechanisms of the tumor:
First, viruses can take advantage of the same pathways that tumor cells activate during malignant progression, for their own growth—resulting in destruction of the tumor (Bergmann M, et al., 2001, Cancer Res., 61: 8188-93; Muster T, et al., 2004, Int J Cancer 110: 15-21; Kim, et al., 2010, Oncogene 29: 3990-3996; Mansour M, et al., 2011. J. Virol. 85: 6015-6023).
Second, viruses are capable to activate both innate and adaptive immune responses against the tumor (Prestwich, R J et al., 2009, Clin Cancer Res 15: 4374-4381; Kim et al., 2010; Immunol. Letters, 134(1), November 30; 134(1):47-54, Ramirez et al., 2010, Discov Med 10: 387-393).
Importantly, these inherent immunogenic properties of the virus can be further enhanced by introducing immunostimulatory molecules such as cytokines and tumor associated antigens into the virus.
Viruses as “dual mechanism cancer therapies”—both killing cancer cells and inducing anti-tumor immune response—represent one of the most promising new strategies to treat cancer. Virotherapy reduces the bulk of the tumor and modulate the immunosuppressive environment by activation of toll-like receptors and expression of transgenic immune enhancing cytokines. The immune enhancing cytokines activate and stimulate cancer-specific T-cells, which subsequently eliminate residual and metastatic tumor cells that may be resistant to viral lysis. It has become increasingly clear that the innate and adaptive immune responses triggered by oncolytic viruses in an otherwise immunosuppressive environment of a tumor are critical components of the clinical benefit of these therapeutics. The potential to modulate the immune suppressive environment of the tumor is due to the inherent ability of many viruses to be strong inducers of T-cell mediated immune responses: T-cell numbers in the body are maintained at a homeostatic steady state unless disturbed by infection or lymphopenia. Inflammatory responses to most pathogens result from the recognition of pathogen-associated molecular patterns by receptors on innate immune system cells like dendritic cells (DC) and natural killer (NK) cells. For example, toll-like receptors recognize structures unique to pathogens such as double stranded RNAs, and toll-like receptor ligation signals the production of cytokines and chemokines that recruit and induce expansion of T cells specific for the infecting pathogen. In contrast to tumor cells which do not express pathogen-associated molecular patterns and therefore fail to activate the innate immune system, most viruses encode several toll-like receptor ligands that effectively activate innate immunity. In particular RNA viruses are strong inducers of innate immune responses since they generate double stranded RNA during replication which effectively interacts with toll-like receptors (Diebold et al., 2003, Nature 424: 324-8.; Shi, Z, et al., 2011, J Biol Chem 286: 4517-4524; Ahmed, M, et al., 2009, J Virol 83: 2962-2975; Appledorn et al., 2011, Clin Vaccine Immunol 18: 150-160). As a consequence, upon intratumoral delivery the mere presence of a virus within a tumor can act as a “danger signal” to alert and activate the immune system (Gallucci and Matzinger, 2001, Curr Opin Immunol 13: 114-119).
The oncolytic properties of influenza virus with deletions in the NS1 gene and their lack of replication in normal cells were reported. However, the mutants were limited to tumor cells with defects in the interferon pathway (WO2009/007244A2). The mutants described in WO2009/007244A2 are characterized by a complete lack of a functional RNA-binding site.
Other mutants not limited to tumor cells with defects in the IFN pathway were only slightly attenuated (WO2004111249A2). Effective replication and expression of the heterologous gene by the vector described in WO2004111249A2, was reported not to be limited in IFN competent cells or sensitive to the effects of IFN.
However, the vector of WO2004111249A2 also grows effectively in normal (IFN competent) cells and animals. Importantly, in contrast to the vector described in the present invention it does not have an optimal conditional replication phenotype. Therefore it does not fulfil an important requirement for a virotherapy approach.
The influenza virion consists of an internal ribonucleoprotein core (a helical nucleocapsid) containing the single-stranded RNA genome, and an outer lipoprotein envelope lined inside by a matrix protein (M1). The segmented genome of influenza A and B virus consists of eight segments, seven for influenza C, of linear, negative polarity, single-stranded RNAs which encode eleven, some influenza A strains ten, polypeptides, including the RNA-dependent RNA polymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which form the nucleocapsid; the matrix membrane proteins (M1, M2 or BM2 for influenza B, respectively); two surface glycoproteins which project from the lipid containing envelope: hemagglutinin (HA) and neuraminidase (NA); the nonstructural protein (NS1) and the nuclear export protein (NEP). Influenza B viruses encode also NB, a membrane protein which might have ion channel activity and most influenza A strains also encode an eleventh protein (PB1-F2) believed to have proapoptotic properties. Transcription and replication of the genome takes place in the nucleus and assembly occurs via budding on the plasma membrane. The viruses can reassort genes during mixed infections. Influenza virus adsorbs via HA to sialyloligosaccharides in cell membrane glycoproteins and glycolipids. Following endocytosis of the virion, a conformational change in the HA molecule occurs within the cellular endosome which facilitates membrane fusion, thus triggering uncoating. The nucleocapsid migrates to the nucleus where viral mRNA is transcribed. Viral mRNA is transcribed and processed by a unique mechanism in which viral endonuclease cleaves the capped 5′-terminus from cellular heterologous mRNAs which then serve as primers for transcription from viral RNA templates by the viral transcriptase. Transcripts terminate at sites 15 to 22 bases from the ends of their templates, where oligo(U) sequences act as signals for the addition of poly(A) tracts. Of the eight viral RNA molecules of influenza A virus so produced, six are monocistronic messages that are translated directly into the proteins representing HA, NA, NP and the viral polymerase proteins, PB2, PB1 and PA. The other two transcripts undergo splicing, each yielding two mRNAs which are translated in different reading frames to produce M1, M2, NS1 and NEP. In most of influenza A viruses, segment 2 also encodes for a second protein (PB1-F2), expressed from an overlapping reading frame. In other words, the eight viral RNA segments code for eleven proteins: nine structural and 2 non-structural (NS1, PB1-F2) proteins.
There is a constant and unmet need for virotherapy which effectively destroys a wide variety of tumor cells and tumors but is sufficiently attenuated in normal cells or tissues.